Our long-term objective is to understand the mechanisms that control the differentiation of specific cell types in the pancreas. Our general strategy is to focus on the beta cell as a model for differentiation in the pancreas. Starting with the insulin gene as an example of a differentiated gene product, we have worked backwards to describe and understand the transcription factor network that controls the differentiation of beta cells from pluripotent progenitor cells. Now we are focused on identifying and placing factors within this network and understanding how these factors guide progenitor cells to differentiate into beta cells. Our specific aims are directed toward dissecting the role of a single factor, the homeodomain transcription factor Nkx2.2, in this transcription factor network. We have chosen Nkx2.2 because it plays a critical role in beta-cell differentiation and because it is uniquely expressed in both early and late progenitor cells, via distinct mechanisms: I. Determine how the expression of Nkx2.2 is differentially directed to progenitor cells and mature beta-cells. Specific targeting of the Nkx2.2 lb promoter to ngn3+ progenitor cells serves as a model of progenitor-specific gene expression. II. Trace the lineage of early and late progenitor cells expressing Nkx2.2 by uniquely labeling the progeny of these cells by cre-mediated recombination of a floxed marker gene. III Test the role of Nkx2.2 in early and late progenitor cells by rescuing Nkx2.2 null embryos with early and late expressing Nkx2.2 alleles. IV. Identify genes downstream of Nkx2.2 in cultured cells infected with an adenovirus expressing Nkx2.2. These studies will help us understand how the differentiated state is established and maintained. In addition, the focus on the beta cell has particular relevance to diabetes. A better understanding of how beta-cells differentiate will help us develop methods for producing beta-cells for patients with diabetes.